Sc 7269

The renal cortexices from the diabetic and non-diabetic kidneys were lysed with protease and phosphatase inhibitors to prevent protein degradation and stored at −80 °C until analysis. Fifty microgram of total protein lysates were denatured, resolved using 10% SDS-PAGE, and transferred to a nitrocellulose membrane, as described in detail previously [18 (link)]. The membrane was blocked with 5% dry milk and incubated overnight with rabbit anti-CX43 antibody at 1:6000 (#C6219, Sigma), or anti-VEGFA at 1:200 (#sc-7269; Santa Cruz Biotechnology, TX, USA). After incubation with a horseradish peroxidase–conjugated secondary IgG (Sigma), the blots were developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate (#34095; Thermo Scientific, MI, USA). Chemiluminescent signals were captured using an ImageQuant LAS 4000 Imager (GE Healthcare Bio-Sciences AB, Sweden) and analyzed by ImageJ software (http://imagej.nih.gov/ij/download.html). Ponceau S staining was used as the loading control.

Standard Western blot analysis [4 (link)] of cerebellum lysates (n = 6/time interval, n = 18 control, and n = 18 PNV treated per age) was performed using rabbit polyclonal antibody against CaB (1 : 2000, C2724, Sigma-Aldrich, St. Louis, MO, USA), Flt-1 (1 : 500, sc-316), Flk-1 (1 : 250, sc-315), both from Santa Cruz Biotechnology (Santa Cruz, CA, USA), GAD (1 : 1000, AB1511, Millipore, Billerica, MA, USA) and laminin (1 : 500, L9393, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal antibody against VEGF (1 : 500, sc-7269) and β-Catenin (1 : 600, sc-7963), both from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and β-actin (1 : 1000, A2228, Sigma-Aldrich, St. Louis, MO, USA) and goat monoclonal antibody against occludin (1 : 500, sc-8144, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were visualized using chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). For quantification, the density of pixels of each band was determined by the NIH Image J 1.45s software (available at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, NIH, Bethesda, MD). For each protein investigated the results were confirmed in three sets of experiments and data were normalized using the respective loading controls. Values were normalized to the corresponding value for β-actin internal control and expressed as a ratio.

Lung tissues were homogenized in a cold buffer consisting of a mixture of protease inhibitors, 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylene glycol tetraacetic acid, and 1 mmol/L ethylenediaminetetraacetic acid (entire mini tablets; Roche, Mannheim, Germany). A 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve proteins (30 g) under reduced conditions, and the proteins were electroblotted to a polyvinylidene difluoride membrane (Immobilon P, Millipore). After being blocked with 5% non-fat dry milk, membranes were incubated with anti-β-actin (1:1000; C4 sc-47778, Santa Cruz Biotechnology) and anti-GPX4 (1:750; SC-7269, Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated goat anti-mouse (Pierce Biotechnology, Rockford, IL, USA). The protein bands were identified with a Pierce BioSpectrum AC System. The intensity of the TOMM20 and β-actin bands on AIDA was measured using densitometric analysis. After normalizing with β-actin, the densitometry unit of TOMM20 protein expression in the RA + NS group was designated as 1.

HBMVECs and brain tissues were harvested and lysed in RIPA lysis buffer (Sigma-Aldrich). The lysate was centrifuged at 11,000 ×g for 2 min at 4 °C to collect the proteins. The proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, pre-cut size was 6.6 × 8.5 cm). The membranes were blocked with 5% skim milk for 2 h and hybridized with primary antibodies to VEGF (1:1000, sc-7269, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), PI3K (1:2000, ab86714, Abcam, Cambridge, UK), p-PI3K (phospho Y607, 1:1000, ab182651, Abcam), AKT (1:800, 10176-2-AP, ProteinTech Group, Chicago, IL, USA), p-AKT (1:500, 66444-1-Ig, ProteinTech Group) and GAPDH (1:1000, ab8245, Abcam) overnight at 4 °C. The membrane was washed and incubated with secondary antibodies goat anti-mouse IgG H&L (HRP) (1:5000, ab205719, Abcam) or goat anti-rabbit IgG H&L (HRP) (1:5000, ab6721, Abcam). The blots were then visualized by Novex™ enhanced chemiluminescence reagents (Thermo Scientific), and the results were normalized to GAPDH.

Gastrocnemius and hearts lysates (obtained by centrifugation of homogenates) were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed using antibodies against MyoD, Myogenin (M3512, M3559-Dako), Desmin, Collagen type-III, α-SMA (D1033, C7805, A5228-Sigma-Aldrich), PGC1α, β-ATPase (AB3242, MAB3494-Millipore), TFAM, Tom20, CSQ2, fsTn-I, VEGF, VE-Cadherin, PECAM, SDHA and PPARγ (sc-23588, sc-11415, sc-390999, sc-377382, sc-7269, sc-9989, sc-46694, sc-377302, sc-7273-Santa Cruz Biotechnology), CoxIV (Ab14744-Abcam) and the appropriate secondary antibodies. α-tubulin or actin (T5168, A3853-Sigma-Aldrich) were used for normalization. Quantification was performed as previously described (Ferraro et al., 2016 (link)).